The development of antibiotic-resistant strains of bacteria has stimulated great interest in reducing the unnecessary use of antibiotics. For this to be accomplished, some means for rapidly determining whether the causal agents are viral or bacterial is required. Further, if the best treatment is to be initiated early in infection before results from culture studies are available, the identification must be sufficiently precise to allow the optimal antibiotic or antiviral to be prescribed. Therefore, the first problem for any medical practitioner presented with a patient having symptoms of an infection is to determine whether the cause is a microorganism which can be treated with an antibiotic (bacteria, mycoplasmas, nanobacteria, or yeasts), or is due to a virus. Presently, the medical practitioner determines the type of infectious agent based on symptoms, on a specific test, or on a best guess as to the agent. If no agent is cultured, it is deduced that the infectious agent is probably a virus. Viruses are also detected and characterized using infectivity assays in which cytopathic effects are observed. Since different viruses can and do infect different cell types, infecting a variety of different cells in culture and determining which are infected makes identification feasible. Once culture conditions are found in which a virus grows, batteries of species or/or strain-specific antibodies are used to determine which one will inhibit infection, thus providing more certain identification. Tissue culture-based virus identification, while definitive if they are positive, are time-consuming and costly, and rarely provide information in time to affect therapy. Hence, current interest in developing more rapid detection and identification methods.
Virus particles have been detected using sandwich immunoassays in which antiviral antibodies are immobilized on solid phases, such as the wells of microtiter plates, and used to capture viruses from sample suspensions. Once the viruses are immobilized and the capture surfaces are washed, a second set of antibodies are added. These attach to the free viral surfaces, and binding is detected. The second set of antibodies may be directly labeled or indirectly labeled to produce detectable signals. The core problem with sandwich immunoassays for viral diagnosis is sensitivity, particularly when only a small number of viral particles are present in a sample. Even when a large number of infectious particles are present, only a small fraction of them are immobilized because of the very slow rate at which a majority of the virus particles diffuse into contact with the antibody-coated walls of a tube or well. Further, those particles that are immobilized are spread over the entire wall and bottom surfaces thereby further diluting the signals resulting from the binding assay. In most samples from infected patients the concentration of viruses is too small to allow detection by this method without first replicating the viruses.
Methods have also been developed for fluorescently staining viruses with stains that attach to DNA or RNA, and which exhibit increased fluorescence after such binding. Handbook of Fluorescent Probes and Research Chemicals, 6th ed. Molecular Probes, Eugene Oreg. (1995). Thus, viruses have been detected by emission of fluorescent light, either using continuous illumination, or pulsed illumination to detect delayed fluorescence. While individual fluorescently labeled virions are detected and counted using the epifluorescent microscope, such methods have not been developed for routine clinical use, but have been used to determine the titers of virus particles in the ocean. Fuihrman, Nature 399:541-548 (1999).
Currently, viruses are also detected and the titer estimated using the polymerase chain reaction (PCR). Tang et al, Clinical Chemistry 43:2021-2038 (1997). PCR requires specific primers, and one complete assay is required for each viral species or type suspected of being present. These assays are relatively fast, require stringent lab conditions, are somewhat expensive, and are currently used to diagnose only a few viruses including cytomegalovirus, and HIV. Furthermore, a single base polymorphism, mutation or variant strain can prevent primer annealing and thereby defeat PCR. The chief difficulty with PCR in the clinic, however, is that one must have some intuition as to the identity of the virus being tested for, since very specific reagents are required, and it is too costly to run a large number of PCR tests on each of many samples.
Affinity chromatography has been used to isolate viruses or their antibodies but these require prior knowledge of one or the other. See Lecomte et al, Journal of Immunological Methods, 13:355-365 (1976) and Kenyon, Science 179: 187-189 (1973).
Using the above methods, viral loads have been determined for a number of viruses including hepatitis B, HIV, and cytomegalovirus. Bai et al, Science 272:1124-1125 (1996). However, for the majority of human viruses, very little quantitative data on the number titer of circulating infectious or physical particles as a function of stage of disease is available. Titers as high as 108 particles/ml have been reported for hepatitis B, while for many infectious diseases, titers based on infectivity have been as low as 103 mL.
Low titers based on infectivity may be due to complexing of virions with antibodies to produce immune complexes, or to rapid removal of these agents by lymphocytes. Experimentally immune complexes containing infectious particles have been widely observed during virus infections. Since these complexes include antibodies specific for the virus involved, they also offer opportunities for the development of diagnostic methods for virus diseases. First binding the immune complexes followed by extraction of the virus per se has isolated viruses (or their antigens). See Zalan et al, Archiv Fur die gesamte Virusforschung 42:307-310 (1973), Snyder et al, Journal of Immunology 128:2726-2730 (1982), McDonald, Immunology 45: 365-370 (1982), Gazitt et al, Immunology Letters, 11:1-8 (1985), Cafruny et al, Infection and Immunity, 37:1001-1006 (1982), Birkbeck et al, Immunochemistry 8:1029-1039 (1971). Antibodies have also interfered with virus recovery, Burger et al, Am. J. Vet. Res. 44:86-90 (1982) and neutralization of viruses, Massey et al, Science 213:447-449(1981).
Means have been developed for amplifying fluorescent signals from immobilized antigens including viruses. These include the use of large fluorescent particles to which are attached virus specific antibodies. The large particles include fluorescent latex beads, dendrimers of branching DNA which forms a scaffold to which are attached both specific antibodies and fluorescent dyes, phage particles displaying specific antibodies (Winter et al, Annual Review of Immunology 12:433-455), and other techniques known to those skilled in the art.
In current medical practice, only a few types of viral infection are routinely observed in a local patient population at any time. Most common are rhinovirus infections and influenza. The rapid identification of the virus is important in order to begin appropriate antiviral therapy or public health measures, if any. However, it is also important to develop tests for minor viral diseases of wide occurrence, for rare and especially fatal viruses, and for new agents that may or may not be agents of biological warfare or terrorism.
Conventional rapid detection systems for viruses, such as infectivity, immunoassays and nucleic acid based assays, require specific prior knowledge of virus. Generally an antibody from convalescent serum of a patient or generated by artificial immunization is needed for an immunoassay. Not all organisms, cells and fragments therefrom induce production of antibody naturally. Likewise, nucleic acid probes and amplification primers require previous knowledge of or sequencing of at least part of the viral genome. Infectivity assays require knowledge of which cell line(s) to use and optionally which interfering or enhancing viruses to use. These tests assume that the agent tested for has been previously isolated and characterized, and that specific reagents are available.
Physical methods for virus counting have depended on a pre-separation to remove contaminating particles, which are generally larger, or have different buoyant densities than viruses, followed by centrifugation onto electron microscope grids. The detection limits of such methods is generally about 105 per ml.
While individual virions may be detected by electron microscopy or epifluorescent microscopy using purified preparations, there has been no general clinically useful methods for diagnosing which, of a small currently circulating set of viruses, a given patient has. Further, no generally useful diagnostic method has been available to identify viruses when they are present in immune complexes,
Blood cell typing has been performed by incubating erythrocytes with antibody and centrifuging the complex in a container with a conical or keel-shaped bottom recess which was previously coated with antibody binding agents (anti-Ig or protein A). The blood type was determined by the amount of sediment formed at the bottom of the centrifuge tube. See Stocker, U.S. Pat. No. 4,560,647. It was proposed to detect viral particles in a manner similar to blood cells, but the concept was not actually performed.
Therefore a great need exists for rapid and general methods of identifying viruses which can apply to all of them, and which does not require specific reagents, special cultures or any preconception of what the virus is.